Abstract 3267: <i>ARID1A</i> mutated bladder cancer cells are sensitized to BET protein inhibition with OTX-015
Authors: Ryan M. Kemper, Heemaja Mewada, Jeffrey S. Damrauer, Brian Hardy, Stephen F. Frye, Kenneth H. Pearce, Jesse Raab, William Y. Kim, Daniel James Crona
Journal: Cancer Research (2022)

Abstract Background: Bladder cancer (BC) is a common and deadly disease. Inactivating ARID1A mutations occur in up to 30% of metastatic BC tumors, making it the most commonly mutated epigenetic gene and the 4th most commonly mutated gene overall in BC. ARID1A mutations are associated with decreased response to therapy and poor prognosis; thus, there is a need to develop therapies specifically targeting ARID1A mutant tumors. Using the UNC EpiG Diamond compound library, a set of well-annotated small molecule probes targeting chromatin regulatory proteins, we determined that inhibitors of bromodomain and extraterminal (BET) proteins potently inhibit the viability of BC cells. Here, we tested the hypothesis that ARID1Amut cells are particularly sensitized to the pan-BET inhibitor OTX-015. Methods: AR1D1A-competent (ARID1AWT) 5637 and ARID1A-mutated (ARID1Amut) HT1197 cells were treated with eight ascending concentrations of OTX-015 (0.1 nM-100 µM), and cell viability was measured using CellTiter-Glo™ after 72-120-hour incubations. IC50 values were calculated using a four-parameter non-linear regression model. Gene expression of BET inhibitor target genes, MYC, ARID1B, and RAD51, were evaluated by RT-PCR after a 48-hour incubation, normalized to an SDHA housekeeper and compared to a 0.1% DMSO control. CellTiter-Glo™ and RT-PCR experiments were conducted in technical and biologic triplicates. Western blotting evaluated OTX-015 effects on c-MYC, BAF250B (encoded by ARID1B), and RAD51 protein expression after 72-hour incubation. Results: OTX-015 was 8-times more potent in ARID1Amut HT1197 cells at 120 h than in ARID1AWT 5637 cells (IC50: 0.12 μM vs. 1.0 μM). OTX-015 treatment (1 µM) significantly reduced ARID1B and RAD51 mRNA expression versus 0.1% DMSO control (83% and 86% reduction, respectively, both P&amp;lt;0.0001). At the same concentration, OTX-015 did not significantly reduce MYC mRNA expression HT1197 cells (25% reduction, P=0.31), but did significantly reduce MYC expression in 5637 cells (57% reduction, P&amp;lt;0.0001) at 48 h. When comparing HT1197 to 5637 cells, OTX-015 treatment (1 µM) caused a greater reduction in ARID1B mRNA expression (83% vs. 62% reduction, P=0.02) and RAD51 mRNA expression (86% vs. 57%, P=0.001) at 48 h. Finally, OTX-015 treatment (1 µM) also resulted in more dramatically reduced c-MYC, ARID1B and RAD51 protein expression in HT1197 cells at 72 h, when compared to 5637 cells. Conclusions: These preliminary results support future lines of inquiry into the molecular mechanisms that underlie sensitization of ARID1Amut cells to BET protein inhibition. Citation Format: Ryan M. Kemper, Heemaja Mewada, Jeffrey S. Damrauer, Brian Hardy, Stephen F. Frye, Kenneth H. Pearce, Jesse Raab, William Y. Kim, Daniel James Crona. ARID1A mutated bladder cancer cells are sensitized to BET protein inhibition with OTX-015 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3267.